Lipase (EC 3.1.1.3) is able to catalyze a wide range of chemical reactions, which include nonspecific and stereo-specific hydrolysis, esterfication, trans-esterification, and interesterification. In addition, it catalyzes the hydrolysis of an ester bond at water-lipid interface. See, e.g., Ader et al. (1997) Methods Enzymot. 286: 351–385; Gandhi (1997) J Am Oil Chem. Soc. 74: 621–634; Klibanov (1990) Acc. Chem. Res. 23: 114–120; Shaw et al. (1990) Biotechnol. Bioeng. 35: 132–137; and Wang et al. (1988) Biotechnol. Bioeng. 31: 628–633.
Due to its catalytic abilities, a Candida rugosa lipase, among commercial lipases, is widely used in bioindustries. Generally, crude C. rugosa lipases are applied in almost all biocatalytic applications, however, enzymes from various suppliers have been reported to show variations in their catalytic efficiency and stereospecificity. See Barton et al. (1990) Enzyme Microb. Technol. 12: 577–583. Several lipase isomers (i.e., isozymes) have been isolated from the crude C. rugosa lipase, and the lipase isozymes were shown to be different in catalytic efficiency and specificity. See Shaw et al. (1989) Biotechnol. Lett. 11: 779–784; Rúa et al. (1993) Biochem. Biophysl Acta 1156: 181–189; Diczfalusy et al. (1997) Arch. Biochem. Biophys. 348: 1–8.
To date, five lipase-encoding genomic sequences from C. rugosa have been characterized. See, for example, Longhi et al. (1992) Biochim. Biophy. Acta 1131: 227–232; and Lotti et al. (1993) Gene 124: 45–55. The five lipase-encoding genes (LIP1, 2, 3, 4, and 5) have been isolated from a SacI genomic library of the yeast C. rugosa by colony hybridization. The five genes encode for mature proteins of 534 residues with putative signal peptides of 15 (in LIP1, 3, 4, and 5) and 14 (in LIP 2) amino acids in length, respectively. The five deduced amino acid sequences share an overall identity of 66% and similarity of 84%. Due to a high sequence homology among the five deduced amino acid sequences and the differential expression level of the five lipase genes (Lee et al. (1999) Appl. Environ. Microbiol. 65: 3888–3895), it is difficult to purify each isozyme directly from the cultures of C. rugosa on a preparative scale for industrial applications.
Further, although these isozymes are conserved at a catalytic triad (including amino acids S209, H449, and E341) and at the sites involved in disulfide bonds formation (including amino acids C60, C97 and C268, C277), they differ in N-glycosylation sites, isoelectric points, and some other features in their hydrophobic profiles. In addition, each of the isozymes may account for certain properties, such as catalytic efficiency and specificity. See Chang et al. (1994) Biotechnol. Appl. Biochem. 19: 93–97. Accordingly, cloning and functional expression of a C. rugosa lipase isozyme are desirable for producing a pure isozyme with certain properties for industrial applications.
However, C. rugosa is a dimorphic yeast in which the triplet CTG, a universal codon for leucine, is read as serine. As a result, the functional expression of a C. rugosa isozyme becomes unfeasible in a common host cell (in which CTG is read as leucine). See Kawaguchi et al. (1989) Nature 341: 164–166.